Animal model system for squamous cell carcinoma

ABSTRACT

Non-human mammalian animals having a higher epidermal expression level of protein kinase Cε than their wild-type counterparts are phenotypically distinguished from wild-type animals in that the animals induced to develop tumors in a chemical initiation/promotion protocol are suppressed for subsequent papilloma development but are susceptible to developing squamous cell carcinoma and metastatic squamous cell carcinoma. The animals are advantageously used in methods for screening putative agents for altering the susceptibility, development and progression of squamous cell carcinoma and metastatic squamous cell carcinoma and have further commercial value as tools for investigating the development of metastatic disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

Not applicable.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with United States government support awarded bythe following agencies:

NIH CA35368.

The United States has certain rights in this invention.

BACKGROUND OF THE INVENTION

A majority of human cancers originate from epithelial tissue. A commoncancer of epithelial origin is nonmelanoma skin cancer (NMSC), includingbasal cell carcinoma (BCC) and squamous cell carcinoma (SCC), with morethan 700,000 new cases diagnosed each year in the United States. Similarcancers are also seen in non-human animals such as domesticated animalsand pets, including cats and dogs. BCC is rarely life-threateningbecause it is slow growing and is mostly localized. Unlike BCC, SCCmetastasizes at a rate of 2% to 6% over several years after initialdiagnosis. A highly malignant form invades and destroys tissue, and thenmetastasizes, initially to a regional lymph node before more distantorgans such as the lung or brain are affected. SCC is commonlyencountered in a number of epithelial tissues, including the oralcavity, esophagus, larynx, bronchi, intestines, colon, genital tract,and skin. Early detection using reliable biomarkers is desired, as arerationally designed drugs for effectively preventing and treatingaggressive, metastatic SCC.

As such, there is a need for a good animal model system for studying howmetastatic squamous cell carcinoma develops, progresses and can betreated. To date, no such model exists. Classically, tumor cells areinjected into the tail vein of either immunocompromised or syngeneicmice. While this assay can suitably model the later metastatic stages,it does not model the early genesis, invasion and angiogenic stages ofmalignant progression, especially as it relates to complex interactionsbetween tumor and host, especially at the tissue site where thecarcinoma originated. Moreover, the role of the immune system inmetastatic progression cannot be analyzed when immunocompromised miceare used.

Murine skin model systems are still essential contributors to ourunderstanding of the multi-step nature of chemically-inducedcarcinogenesis. In the multistage mouse skin carcinogenesis model,biochemical events unique to initiation, promotion, or progression canbe studied and related to cancer formation. In that model, the NMSC thatis most often induced is squamous cell carcinoma. Although squamous cellcarcinoma of mouse skin invades the dermal region, the incidence ofmalignant metastatic conversion is rare and requires a long latencyperiod of approximately a year.

Several protocols are used to develop mouse skin tumors in laboratoryanimals. In a common initiation-promotion protocol, mouse skin istreated with an initiating agent (7,12-dimethylbenz[a]anthracene; DMBA)and then with a potent tumor promoter(12-O-tetradecanoylphorbol-13-acetate; TPA). In this protocol, micedevelop mostly benign papillomas, more than 90% of which regress afterTPA treatment is stopped. Only a small percentage of papillomas progressto invasive, but non-metastatic, SCC. The initiation-promotion protocolhas been further modified to enhance the conversion of skin papillomasto carcinomas, yet metastatic potential is not increased.

A major intracellular receptor for TPA is the ubiquitous enzyme proteinkinase C (PKC), an important signal transduction pathway component forcontrolling cell proliferation and tumorigenesis. It has been suggestedthat PKC activation may play a role in promoting mouse skin tumorformation. However, several groups have demonstrated that repeatedapplications of TPA depress PKC activity and protein levels. Theseresults indicate that both loss of PKC activity and degradation of PKCcould be important for mouse skin tumor promotion by TPA.

On the basis of the structural similarities and cofactor requirements,the eleven known PKC isoforms are grouped into three subfamilies: (1)the conventional PKCs (α, βI, βII, and γ), which depend upon Ca²⁺,phosphatidylserine (PS), and diacylglycerol (DAG) or TPA; (2) the nPKCs(ε, δ, η, and θ), which require only PS and DAG/TPA; and (3) theatypical PKCs (ι/λ and ξ), which retain PS dependence but have norequirement for Ca²⁺ or DAG/TPA for activation. PKCμ, which is usuallyclassified as a nPKC, is not easily grouped with any of the otherisoforms.

The roles of PKCα and PKC_(δ) isoforms in the mouse skin tumorinitiation/promotion protocol were assessed in FVB/N transgenic miceexpressing an T7-epitope-tagged PKCα (T7-PKCα) or PKCδ (T7-PKCδ) underthe control of the human keratinocyte-specific K14 promoter/enhancer.Transgenic expression of T7-PKCα did not affect tumor promotionsusceptibility. Transgenic expression of T7-PKCδ in the epidermis(˜8-fold increase) suppressed the formation of both skin papillomas andcarcinomas by 70%.

PKCε may play an important role in cellular growth regulation. TPA bindsto and activates PKCε. Activated PKCε may be important for the survivalof small cell lung carcinoma cell lines in which the catalytic fragmentof PKCε is constitutively expressed. Overexpression of PKCε in Rat-6 orNIH-3T3 fibroblasts increases growth rate, anchorage independence, andtumor formation in nude mice. PKCε overexpression also transformsnon-tumorigenic rat colonic epithelial cells and suppresses apoptosis ofinterleukin-3 dependent human myeloid cells induced by removal ofinterleukin-3.

The role of PKCε in mouse skin tumor promotion and epidermal cell growthand differentiation remains unclear. Treatment of the mouse skin withTPA leads to a general reduction in PKC activity that persists for atleast 4 days. Acute TPA treatment decreases PKCβ and η protein levels,but has little or no effect on the levels of PKCα, δ, or ε. PKCα, β, andδ activity levels were reduced after acute or repeated TPA treatments,but PKCε activity was not examined. DMBA/TPA-induced papillomas exhibitdecreased cytosolic levels of PKCα and βII protein, but insignificantalterations in the levels of PKCδ, ε, or ξ protein. When cultured mouseskin keratinocytes are induced to differentiate by increasing Ca²⁺,PKCε, δ, and a translocate to the membrane fraction, suggesting a rolefor activation of these isoforms in keratinocyte differentiation.

BRIEF SUMMARY OF THE INVENTION

The present invention is summarized in that an FVB/N mouse thatexpresses more PKCε in its epidermis than in the epidermis of awild-type FVB/N mouse is a useful model for development and treatment ofskin cancer, particularly squamous cell carcinoma, in human andnon-human mammalian animals. In a preferred embodiment, the mouseexpresses at least about 5-fold more epidermal PKCε than wild-type FVB/Nmice, with more preferred embodiments having still higher levels ofepidermal PKCε.

The present invention is also summarized in that an FVB/N mouse thatexpresses PKCε in its epidermis at a level higher than a wild-type FVB/Nmouse, where the level is sufficiently high to induce metastatic growth,is a useful model for development and treatment of metastatic skincancer, particularly for metastatic squamous cell carcinoma, in humans.Notably the mice constitute a model system for developing and treatinghighly malignant metastatic squamous cell carcinoma. A levelsufficiently high is more than 5-fold higher than in wild-type FVB/Nmice, and is preferably at least about 12-fold higher, and still morepreferably at least about 15-fold higher, and most preferably at leastabout 18-fold higher.

The present invention is also summarized in that a method for inducingsquamous cell carcinomas in the aforementioned mice includes the stepsof treating the mouse with a skin tumor initiating chemical agent, thentreating the mouse repeatedly with an skin tumor promotion chemicalagent for a time sufficient to induce squamous cell carcinomas and thenscreening the treated mice to identify those mice in which squamous cellcarcinoma is induced. In a related embodiment, the skin tumor initiatingagent can be DMBA and the skin tumor promotion agent can be TPA. Aftertreatment according to the method, the mice of the invention arecharacteristically suppressed for papilloma formation, even thoughsquamous cell carcinoma is observed at an enhanced rate. The mice havingsquamous cell carcinoma disease produced in the inducing method are alsoan useful animal model for development and treatment of squamous cellcarcinoma induced by non-chemical agents such as ultraviolet radiation.

In a related aspect, the present invention is further summarized in thata method for inducing squamous cell carcinomas in FVB/N mice thatexpress PKCε in its epidermis at the aforementioned level sufficientlyhigh to induce metastatic growth consists essentially of the step oftreating the mice with an initiating agent without further treatmentwith a promoting agent.

In a related embodiment, the present invention is summarized in that amethod for inducing metastatic moderately differentiated squamous cell(MDSC) carcinomas in an FVB/N mouse that expresses at least about12-fold more PKCε in its epidermis than a wild-type FVB/N mouse includesthe steps of treating the mouse with a skin tumor initiating agent andthen treating the mouse repeatedly with an skin tumor promotion agentfor a time sufficient to allow metastatic involvement, typically, butnot exclusively, of the lymph nodes. The metastatic MDSC thus inducedappear to originate from the hair follicle within squamous cells locatednear the sebaceous gland (“bulge region”) that are postulated to beprogenitor or stem cells for the hair follicle and epidermis. Themetastatic MDSC are pathologically distinguishable by histogenesis fromthe well differentiated squamous cell carcinomas (WDSC) observed inFVB/N mice having a wild-type PKCε level that appear to originateinstead from the interfollicular epidermis and which invade the dermisand subcutaneous tissues, but remain localized.

In a related aspect, the invention is summarized in that a method forevaluating the effectiveness of putative agents against squamous cellcarcinoma in a mammal includes the steps of administering various dosesof at least one putative agent over an appropriate range to mice of theinvention that have squamous cell carcinoma, evaluating the effect ofthe agent on development or progression of the squamous cell carcinoma,and selecting at least one agent having anti-squamous cell carcinomaactivity.

The invention is still further summarized in that a method forevaluating the effectiveness of putative agents against metastaticsquamous cell carcinoma in a mammal includes the steps of administeringat least one putative agent in varying amounts to mice of the inventionhaving metastatic squamous cell carcinoma, evaluating the effect of theagent on development or progression of the metastatic squamous cellcarcinoma, and selecting at least one agent having anti-metastaticsquamous cell carcinoma activity.

The present invention is also summarized in that a mouse that expressesat leas about 5-fold more PKCε in its epidermis than a wild-type FVB/Nmouse can be made by increasing the rate at which a PKCε-encodingpolynucleotide is transcribed, by decreasing the rate at which atranscript of the PKCε-encoding polynucleotide is degraded, or byincreasing the stability of the transcript or of a resulting PKCεprotein in epidermal cells.

In a related aspect, the present invention is summarized in that amethod of making a mouse that expresses at least about 5-fold more PKCεin its epidermis than a wild-type FVB/N mouse includes the steps ofintroducing into a one cell fertilized FVB/N embryo a chimeric transgenethat comprises a polynucleotide that encodes PKCε under thetranscriptional control of an upstream promoter active in keratinocytesand a downstream polyA addition sequence, implanting the embryo in acarrying animal, screening progeny of a cross between offspring of thecarrier animal and FVB/N mice for expression of the transgene for PKCεexpression level, and selecting transgenic offspring having increasedPKCε expression.

The present invention is yet further summarized in that a transgene foruse in the method of making a mouse that expresses at least about 5-foldmore PKCε in its epidermis than a wild-type FVB/N mouse comprises apolynucleotide that encodes PKCε under the transcriptional control of anupstream promoter active in keratinocytes and a downstream polyAaddition sequence, and optionally includes a polynucleotide that encodesa peptide tag adjacent to the polynucleotide that encodes PKCε andfurther optionally includes one or more other transcription enhancingelements.

It is an object of the invention to provide a non-human animal modelsystem for cutaneous squamous cell carcinoma and metastatic squamouscell carcinoma in other human and non-human mammalian animals.

It is another object of the invention to provide an animal model systemwhere the non-human animal develops an aggressively malignant andmetastatic disease.

It is a feature of the present invention that carcinomas induced in themice of the invention in a two-stage tumor initiation/promotion methodsuppress the formation of skin papillomas and enhance the formation ofmoderately differentiated squamous cell carcinomas.

It is another feature of the present invention that when the level ofPKCε is sufficiently high in mice of the invention, moderatelydifferentiated squamous cell carcinomas of the animals rapidlymetastasize to regional lymph nodes, there by mirroring cutaneousmetastatic squamous cell carcinoma disease in humans and other non-humanmammalian animals.

It is an advantage of the present invention over existing murine modelsystems in that the invention permits study of metastatic development ina timely manner.

It is yet another advantage of the present invention that the carcinogenis administered topically.

It is yet another advantage of the present invention that the carcinomasappear rapidly, within 15 to 25 weeks, thus facilitating its use inscreening for agents that can prevent induction of metastatic SCC and asa model for investigating the genesis and progression of SCC, and themolecular events associated with progression and metastasis.

Other objects, advantages and features of the invention will becomeapparent upon consideration of the following detailed description.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Not applicable.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a murine model system for developmentand progression of squamous cell carcinomas, and more particularly amodel system for development and progression of metastatic carcinomasand methods for making and using mice for evaluating agents formodulating development and progression of metastatic carcinomas, as wellas the mice per se. Certain aspects of this invention are described inReddig, P. J. et al., “Transgenic Mice Overexpressing Protein Kinase Cεin Their Epidermis Exhibit Reduced Papilloma Burden but EnhancedCarcinoma Formation after Tumor Promotion,” Cancer Research 60:595(2000) incorporated herein by reference as if set forth in its entirety.

Mice within the scope of the invention are characterized as FVB/N micethat express PKCε at or above about five times the level seen inwild-type FVB/N mice. The effects that result from elevated epidermalPKCε levels vary as the epidermal level increases. Overexpression ofPKCε in the untreated mouse epidermis leads to phenotypic abnormalities(such as inflamation, hyperkeratosis, hyperplasia, cellular hypertrophy,and ulceration), especially of the skin surrounding the tail base ofolder mice (approximately 7-8 months of age).

Of particular note in the disclosed system is the different character ofthe tumors induced in mice of the invention after a tumorinitiation/promotion regimen. The mice of the invention are suppressedfor papilloma formation and instead develop moderately differentiatedsquamous cell carcinomas (MDSC) without requiring papillomas as aprecursor. The MDSC appear to originate from the hair follicle withinsquamous cells located near the sebaceous gland (“bulge region”). Thebulge cells are thought to be progenitor or stem cells for the hairfollicle and epidermis. Interestingly, after treatment certain mice ofthe invention also develop papilloma-independent squamous cell carcinomathat metastasizes rapidly to the lymph nodes when the activity level ofPKCε in the epidermis is particularly high, say, at least about 12-foldhigher than the wild-type level, or more preferably greater than 15 foldhigher, most preferably about 18 fold higher.

In contrast, wild-type FVB/N mice treated in the same regimen typicallyfirst develop papillomas and then well differentiated squamous cellcarcinomas (WDSC) that invade the dermis and subcutaneous tissues butremain localized. The WDSC largely appear to originate from theinterfollicular epidermis.

The mice and methods of the invention provide a unique opportunity tostudy the origin and events associated with malignant progression (andinterruption thereof) and have direct commercial value to thebiotechnology community as a result. Separately, the mice and methodsprovide a resource with which to evaluate the role of PKCε intumorigenesis. The difference in metastatic potential and the differentorigin of malignancy support the conclusion that T7-PKCεpapilloma-independent carcinomas are pathologically distinct from themouse carcinomas seen in treated wild-type FVB/N mice.

Mice having elevated epidermal PKCε activity levels in accordance withthe invention can be produced by increasing the rate at which aPKCε-encoding polynucleotide sequence is transcribed, by decreasing therate at which the transcript is degraded, or by increasing the stabilityof the transcript or of the resulting PKCε protein in epidermal cells.One suitable and convenient method for increasing the PKCε level inepidermal cells is to provide in the cells a transgene that comprises aknown PKCε coding sequence flanked upstream by a promoter effective inthe target skin cells and downstream by a suitable polyA sequence,preferably from the same source as the promoter sequence. Other elementsthat can be provided on the transgene include transcription enhancingsequences, such as a β-globin intron, or sequences that can aid indetecting the encoded PKCε protein, such as a T7 tag that can be readilydetected using a commercially available horseradishperoxidase-conjugated anti-T7-tag antibody(Novagen, Inc., Madison,Wis.). Preferably, the additional sequences should not negatively affectthe expression or activity of the PKCε protein.

Nucleotide sequences for the components of a suitable transgene areattached in the Sequence Listing. A K14 promoter/enhancer sequence fromhumans is attached as SEQ ID NO:1. A suitable β-globin intron isattached as SEQ ID NO:2. A PKCε encoding polynucleotide sequence frommice is attached as SEQ ID NO:3. At the 5′ end of SEQ ID NO:3 is a T7tag sequence. SEQ ID NO:4 depicts the encoded PKCε polypeptide sequence.SEQ ID NO:5 is a K14 poly A addition sequence from humans. The skilledartisan will appreciate that the functions of the disclosed sequencesare not adversely affected by certain mutations in the aforementionedpolynucleotide and amino acid sequences, including insertions, deletionsor substitutions, including but not limited to changes that result inconservative changes to any resulting amino acid sequence. Moreover,some changes in either the PKCε coding sequence or a regulatory sequencecan increase the PKCε activity in the epidermis and such changes arespecifically contemplated to be within the scope of the invention.

The transgene can be provided to epidermal cells or to cells that matureinto epidermal cells, such as keratinocytes or keratinocyte precursorcells. Most preferably, the transgene is inserted into an early stagefertilized embryo in a method for making transgenic mice. The embryo isallowed to mature in a manner known to the art. Introduction of thechimeric gene into the fertilized egg of the mammal is accomplished byany number of standard techniques in transgenic technology (Hogan etal., 1986, Manipulating the Mouse Embryo: A Laboratory Manual, ColdSpring Harbor, N.Y.). Most commonly, the chimeric gene is introducedinto the embryo by way of microinjection. The PKCε-encodingpolynucleotide sequence under the control of the strong heterologouspromoter can conveniently be introduced into the recipient mouse strainby microinjection of a chimeric expression cassette (or “transgene”)into pronuclei, preferably male pronuclei, of 1-cell fertilized mouseembryos using known transgenic method as described below in connectionwith the working embodiment of the invention. Other methods, includingclassical breeding methods, can also be used to produce animalsexpressing elevated levels of PKCε in the skin of the animal.

It is important to perform the transgenic technique with a recipientmouse strain that is both susceptible to tumor formation and totransgenic uptake of exogenous DNA. Moreover, the mouse strain should beinbred to eliminate undesired and uncontrollable effects that can beintroduced by heterozygosity. Mice suitable for use in the methods ofthe invention, including the method for producing transgenic mice, areFVB/N inbred mice (commercially available from Taconic Farms, Inc.) andclosely related strains and derivatives thereof, although other mousestrains may also be suitable. The FVB/N inbred mouse strain produceseggs that have easily discernible pronuclei and produce large littersfor an inbred strain. FVB/N mice are also sensitive to multistagecarcinogenesis. See Taketo, M. et al., P.N.A.S. U.S.A. 88:2065-2069(1991), incorporated by reference herein as if set forth in itsentirety.

Animals obtained in the method can be screened for integration of thePKCε-encoding polynucleotide and for production of PKCε in theepidermis. A skilled artisan can use known methods to determine whethera transgene has been incorporated into the genome of offspring mice andcan readily determine the level of expression of the transgene, forexample either by probing for antibodies directed to a tag on thetransgene or antibodies directed to the PKCε protein sequence itself.Preferred methods are described in the Example.

Skin tumors can be induced in the mice using a standardinitiation-promotion regimen wherein a single dose of a tumor initiator(e.g., DMBA) is applied to the backs of the animals. Several weeks afterinitiation, a regimen of applying a tumor promoting agent (e.g., TPA) isundertaken. The time required to observe phenotypic changes in treatedmice will vary somewhat with the mouse strain and with the level of PKCεexpression in the mouse.

The mice of the invention having either squamous cell carcinoma diseaseor metastatic squamous cell carcinoma disease as described as useful forevaluating the effectiveness of one or more putative agents for treatingthe disease. For purposes of this invention, an agent is effective if itreduces the incidence or number of carcinomas on an animal or if reducesthe severity of the carcinomas or if it reduces or eliminates metastaticdisease in those animals in which metastatic disease is observed. In asuitable method for evaluating the effectiveness of a putative agent,the agent, either alone or with a plurality of other putative agents, isadministered topically, orally, intraperitoneally, intramuscularly, orin any other suitable manner to an animal or animals having the diseaseto be treated in an amount to be determined on a case by case basis, buttypically on the order of about 1 mg to about 1 gram (preferably in therange of between about 10 mg and about 100 mg) per mouse having atypical weight of about 25 grams. The effect on the animal of thetreatment is then evaluated and those agents having an effect on thedisease are selected. The agent(s) can have advantageouschemopreventative effect upon the development of metastatic disease inanimals of the invention when administered in the same manner to theanimal before or after development of squamous cell carcinoma in situ(pre-metastatic carcinoma). One can also screen for agents that reduceor reverse the progression of metastatic squamous cell carcinoma inhuman and non-human animals using the mice of the invention as a tester.

As a proof of concept, the inventors have demonstrated that whenchemopreventative difluoromethylornithine (DFMO) was administered orallyin drinking water to mice of the invention at between 50 and 100 mg permouse, squamous cell carcinomas characteristic of the mice of theinvention did not develop after initiation and promotion.

The invention will be better understood upon consideration of thefollowing Example which is to be considered exemplary and not limitingon the scope of the invention

EXAMPLE

Materials and Methods

Materials. TPA and calpain inhibitor I were purchased from AlexisCorporation, San Diego, Calif. DMBA was purchased from Aldrich ChemicalCompany, Inc., Milwaukee, Wis. Proteasome inhibitor Z-Leu-Leu-Leu-H(aldehyde) (MG132) was purchased from Peptide Institute, Inc., Osaka,Japan. Horseradish peroxidase conjugated anti-T7-Tag antibody waspurchased from Novagen, Inc. Madison, Wiss. Rabbit polyclonal antibodiesto PKCε and actin were purchased from Santa Cruz Biotechnology, Inc.Santa Cruz, Calif. DAKO immunoperoxidase LSAB®+Kit was purchased fromDAKO Corporation, Carpinteria, Calif. Immobilized protein A/G agarosewas purchased from Pierce, Rockford, Ill. ECL and ECF Western blottingdetection reagents and the protein kinase C enzyme assay system werepurchased from Amersham, Arlington Heights, Ill. FVB/NTacfBR mice (FVB/Nmice), 7 to 10 weeks of age, were purchased from Taconic Farms, Inc.,Germantown, N.Y.

Generation of transgenic lines. Transgenic mice were produced bymicroinjecting into male pronuclei of one-cell fertilized embryos ofFVB/N×FVB/N mice a T7-tagged expression cassette containing an openreading frame encoding PKCε under the control of the human K14keratinocyte-specific promoter. The K14 promoter has been usedsuccessfully to direct overexpression of several different genes to theepidermis, although other keratinocyte-specific promoters such as the K5promoter can also be used. The protocols for microinjection and forscreening for transgenic animals are described in Reddig, P. J. et al.,“Transgenic Mice Overexpressing Protein Kinase Cε in Their EpidermisExhibit Reduced Papilloma Burden but Enhanced Carcinoma Formation afterTumor Promotion,” Cancer Research 60:595 (2000), already incorporated byreference herein as if set forth in its entirety.

The expression cassette (“transgene”) used in the method was produced asfollows. The Bgl II/Sal I fragment containing the T7 bacteriophageepitope tag open reading frame from the pET-21c(+) vector (Novagen,Inc.) was ligated to the 5′-terminus of the mouse PKCε cDNA in thepRSV-PKCε vector to produce the pRSV-T7-PKCε vector. The T7-PKCε cDNAfrom pRSV-T7-PKCε was ligated into the BamH I site of the pGEM3Z-K14 0β-globin vector by insertion of two Eco47III fragments of the T7-PKCεcDNA, previously linked to Bgl II and BamH I sites, to produce thepGEM3Z-K14-T7-PKCε vector which expressed active T7-PKCε when assayed byimmunocomplex kinase assays of CV-1 cells transiently transfected withthe T7-PKCε vector (data not shown). The K14-T7-PKCε expression cassetteused as the transgene was isolated from pGEM3Z-K14-T7-PKCε by partialdigestion with Hind III and complete digestion with endonuclease Ehe Iand was purified from the contaminating vector DNA by agarose gelelectrophoresis, electroelution, and ion-exchange chromatography. Thecomponents of the preferred transgene are presented in the attachedSequence Listing at SEQ ID NOs:1-3 and 5.

Offspring were analyzed for integration of the K1 4-T7-PKCε expressioncassette. Genomic DNA obtained from biopsies of tail tissues of weanlingmice was digested with EcoRV; the probe was a radiolabelled ˜1kbEcoRV/BamH I fragment of pGEM3Z-K14 β-globin vector containing the K14promoter ligated to the β-globin intron. Nine mice exhibited transgeneintegration after analysis of 24 potential founder mice. These foundermice were bred to wild-type FVB/N mice to produce F1 offspring.Transgenic F1 mice were bred with other transgenic or wild-type FVB/Nmice as necessary to maintain and expand the colony. Mice were housed ingroups of 3-4 in plastic-bottom cages in light, humidity, andtemperature (24° C.) controlled rooms; food and water were available adlibitum. The animals were kept in a normal rhythm of 12-h light and 12-hdark periods.

F1 mice from each transgene positive line were examined by immunocomplexkinase assay for expression of the transgene. The dorsal skin of themice was shaved and depilated 24 h before experimentation. The mice wereeuthanized, the dorsal skin was removed, and the epidermis was scrapedoff on ice with a razor. The epidermis was placed in 0.5 mlimmunoprecipitation (IP) lysis buffer (50 mM HEPES (pH 7.5), 150 mMNaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl₂, 10 μg/ml aprotinin,10 μg/ml leupeptin, 1 mM PMSF, 200 mM Na₃VO₄, 200 mM NaF, 1 mM EGTA, 100mM benzamidine, 5 mg/ml antipain, 5 mg/ml pepstatin, 40 mM MG132, 40 mMcalpain inhibitor I), homogenized using a glass Teflon tissuehomogenizer, agitated for 30 min at 4° C, centrifuged at 14,000 rpm in amicrocentrifuge for 15 min, and the supernatant of the lysate was usedfor immunoprecipitation. The T7-PKCε transgene product wasimmunoprecipitated with the anti-T7 antibody. The lysate waspre-absorbed with 5 82 of protein A/G agarose for 10 min at 4° C. Fiveμg of anti-T7 Tag antibody and 10 μl of protein A/G agarose was added tothe lysate and the volume of the lysate was adjusted to 1 ml with lysisbuffer. The mixture was incubated for 2-4 h at 4° C. with agitation. Theimmunoprecipitate was pelleted at 14,000 rpm in a microcentrifuge,washed, and resuspended in 300 ml assay buffer (50 mM Tris (pH 7.4), 5mM EDTA (pH 8.0), 10 mM EGTA pH 7.9, 0.3% β-mercaptoethanol, 5 μg/mlaprotinin, 5 μg/ml leupeptin, and 50 μg/ml phenylmethylsulfonyl fluoride(PMSF)). The immunoprecipitates were assayed for kinase activity bymeasuring the incorporation of ³²P into an epidermal growth factorreceptor (EGFR) peptide (ERKRTLRRL; SEQ ID NO:6) in the presence ofphosphatidylserine (PS) and TPA. Twenty-five μl of the immunoprecipitatewas assayed in kinase buffer containing 0.2 μCi [γ−³²P ATP], 50 mM Tris(pH 7.4), 8 mM MgCl₂, 0.136 mM ATP, 100 mM EGFR peptide, 3 mM DTT, 34mg/ml of L-phosphatidyl-L-serine, 3 mg/ml TPA and 1 mM EGTA. Thereaction was incubated at 37° C. for 15 min, stopped with 10 ml of 300mM H₃PO₄, spotted onto filter discs, washed with 75 mM H₃PO₄, andcounted. The assay components are commercially available from Amersham.The amount of activated T7-PKCε kinase activity detected in line 215 wasapproximately 19-fold greater than that observed in the line 224 mice.Line 206 displayed constitutive T7-PKCε activity, but exhibited noresponse to the presence of PS and TPA.

Blots probed with the anti-T7 antibody demonstrated that three of theselines were expressing the T7-PKCε transgene. Mouse skin was excised andscraped to remove the subcutaneous tissue. The skin was ground with amortar and pestle under liquid nitrogen. The ground tissue washomogenized with 5 volumes of PKC extraction buffer (20 mM Tris-HCl (pH7.4), 0.3% Triton X-100, 2 mM EDTA, 10 mM EGTA, 0.25 M sucrose, 1 mMDTT, 10 μg/ml leupeptin, and 10 μg/ml aprotinin). The homogenate wascentrifuged at 100,000×g for 60 minutes at 4° C. and the supernatant wasused as the total PKC extract. Protein concentration in the total PKCextract was determined and 100 μg of total PKC extract protein wasfractionated on a 7.5% or 10% SDS-PAGE. The proteins were transferred to0.45 μm supported nitrocellulose membrane. The membrane was thenincubated with anti-T7 Tag (1:2000 dilution) or anti-PKCε (1:100dilution) antibody, the bound antibody was detected using theappropriate secondary antibodies, and the detection signal was developedwith Amersham's ECL or ECF reagents. Immunoblotting of lysates from theimmunocomplex kinase assays was also performed with 100 μg of totalprotein.

Examination of the PKC levels with the anti-PKC antibody demonstratedthat the PKC levels were significantly elevated in these transgeniclines. The PKC protein level was the highest in line 215, whichpositively correlated with the level of PKC activity. The increase inPKC immunoreactive protein levels was 3-, 6-, and 18-fold for line 206,224, and 215, respectively.

The expression pattern of the T7-PKCε transgene was examined in the skinof transgenic and wild-type mice. Staining with the anti-PKCε antibodywas more intense in the dorsal skin of the transgenic mouse than thewild-type littermates. Formalin fixed dorsal skin samples were takenfrom a wild-type mouse and a line 215 T7-PKCε transgenic mouse. Whenhybridized with a polyclonal rabbit anti-PKCε antibody, the wild-typedorsal skin sample exhibited light immunoreactivity throughout theepidermis. Light and infrequent nuclear staining was observed. Stainingof the hair follicles was also observed in the wild-type dorsal skin. Incontrast, PKCε exhibited strong staining in the basal cells of theepidermis and was focally present in the suprabasal layers. Anenhancement of PKCε staining in hair follicle epidermal cells was alsoobserved.

Tissue samples from the dorsal and tail base skin from wild-type andtransgenic mice were examined histologically. These samples were fixedin formalin, sectioned, and stained with hematoxlin and eosin. Alltransgenic mice exhibited several phenotypic alterations with line 215mice exhibiting the greatest penetrance. The mice were phenotypicallynormal at birth. The phenotypic alterations began around 4 to 5 monthsof age for the F1 and F2 mice with a mild hyperkeratosis in the tailepidermis (12 out of 26 mice) around 4 months that did not persist. Thiswas accompanied by persistent inflammation at the base of the tail in 26out of 26 (100%) mice and inflammation of the ears in 20 out of 26 (77%)mice starting about 4 to 5 months of age. The inflammation of the tailbase and ears was followed by the formation of ulcerative lesions atthese sites around 7-8 months of age in 17 out of 26 (65%) transgenicmice observed. Mild, transient inflammation around the eyes was alsoobserved in 7 out of 26 mice (27%) in line 215 T7-PKCε with a variableage of onset. Although not observed in the F1 mice, ulceration in theproximal dorsal skin that became evident at 5 to 6 months of ageoccurred in 7 out of 20 (35%) of the F2 mice. Line 224 and 206 exhibitedphenotypic abnormalities similar to that observed in line 215 mice.However, the severity of the abnormalities was greatly diminished inthose lines.

Histologically, these mice consistently exhibited hyperkeratosis in thedorsal skin, but had no other apparent abnormalities. The grosslyaffected skin regions displayed several significant alterations.Hyperplasia, characterized by acanthosis, was found in the affectedregions along with cellular hypertrophy. Focal regions of necrosis werealso evident at sites of ulceration. Mixed inflammatory cell infiltrateswere observed in the dermal layer with infiltration into the epidermisand keratinizing layers in affected regions. Prominent amongst theinflammatory cell infiltrates were neutrophils and mast cells.

Tumor Promotion. Mouse skin tumors were induced in the mice (line 215)having an 18-fold higher level of PKCε expression than wild type by theinitiation-promotion regimen. At 8-10 weeks of age, the dorsal skins ofthe mice were shaved 3-4 days before treatment, and those mice in theresting phase of their hair cycle were used for experimentation. Themice were initiated by applying 100 nmol of DMBA in 0.2 ml acetone (oracetone alone as a control) topically to the shaved backs. Two weeksafter initiation, 5 nmol of TPA in 0.2 ml acetone (or acetone alone) wasapplied twice weekly to skin for the duration of the experiment. Thenumber of mice for each experimental group was as follows: DMBA-TPA(wild-type females, 11), (transgenic females, 15), (wild-type males,20), (transgenic males, 12); DMBA-acetone (wild-type females, 11),(transgenic females, 16), (wild-type males, 19), (transgenic males, 12);Acetone-TPA (wild-type females, 10), (transgenic females, 15),(wild-type males 19), (transgenic males, 11).

At the beginning of the experiment, the eight to ten week old miceexhibited no phenotypic abnormalities. Tumor incidence and burden wereobserved weekly starting at 4 weeks of TPA promotion. Carcinomas wererecorded grossly as downward-invading lesions, a subset of which wereexamined histologically, and malignancy was confirmed as invading thepanniculus carnosus. Carcinoma bearing mice were killed shortly afterdiagnosis.

Overexpression of PKCε in the epidermis had an unexpected effect onmouse skin tumor promotion by TPA. It had been suggested that PKCε wouldenhance the effects of tumor promotion. In vitro models of cellulartransformation had indicated that PKCε was a potent transforming proteinwhen overexpressed in both fibroblasts and epithelial cells. PKCε aloneinduced complete transformation of these cells, allowing tumor formationwhen these cells were injected subcutaneously in athymic mice. However,a dramatic 95% reduction in the papilloma burden was observed when theline 215 transgenic mice were initiated with DMBA and promoted with TPA.Treatment with DMBA and TPA elicited an average of 20 papillomas permouse in wild-type females and males. In striking contrast, after thesame treatment the male and female transgenic mice averaged fewer than 1papilloma per mouse. For purposes of this invention, an average of fewerthan 1 papilloma per mouse is considered to be no papilloma formation.Moreover, the papillomas in the transgenic mice were small, usually lessthan 2 mm in diameter. Both transgenic and wild-type mice exhibited nodifferences in weight gain during the course of the experiment. At theend of tumor promotion, the total survival was 92% for the wild-typemice and 88% for the T7-PKCε mice.

In spite of the low papilloma burden, the transgenic mice developedcarcinomas independent of papilloma development. After 22 weeks of tumorpromotion with DMBA and TPA, 27% of the female and 50% of the maletransgenic mice developed carcinomas without prior formation ofpapillomas. Although wild-type mice treated with DMBA and TPA alsodeveloped carcinomas by this time (30% and 15% for females and males,respectively), those carcinomas all developed from existing papillomas.Additionally, 25% of the female transgenic mice treated with DMBA alonedeveloped carcinomas by the twenty-first week of promotion.

At the end of the tumor promotion experiment, the T7-PKCε kinaseactivity level in papillomas, carcinomas, and uninvolved epidermis onthe transgenic mice was measured to determine whether formation of thelesions requires modulation of T7-PKCε kinase activity. The kinaseactivity level was determined as described above for the T7-PKCimmunocomplex kinase assay, but with the following modifications. Theskin was not depilated. Skin papillomas and carcinomas were excisedbefore scraping off the uninvolved epidermis. The excised papillomas,carcinomas, and epidermis were separately homogenized and extracted in0.5 to 1.0 ml of IP lysis buffer. For each treatment group, theepidermis from 3 mice were combined for extraction. Two to fourpapillomas or 1-2 carcinomas were excised, combined, and extracted inthe IP lysis buffer. One hundred mg of the total protein extract, priorto immunoprecipitation, was used for immunoblot analysis.

The levels of precipitable T7-PKCε kinase activity in the epidermalextracts were very similar between each of the treatment groups at 72and 120 h after the last TPA treatment. The basal (without PS/TPA) andthe stimulated (with PS/TPA) T7-PKCε kinase activity levels in extractsfrom papillomas or carcinomas from DMBA-TPA treated mice were bothgreatly reduced compared to the levels in the surrounding epidermis.This reduction in kinase activity positively correlated with the reducedlevel of T7-PKCε protein present in the total, epidermal extracts usedfor the immunoprecipitations. Thus, it appears that the elevated levelsof PKCε activity may inhibit papilloma development and this increasedactivity may need to be reduced for papillomas to develop. The lowlevels of immunoreactive PKCε in papillomas from wild-type mice furtherindicates that a reduction in PKCε levels is important for papillomaformation during tumor promotion.

Also surprisingly, in the absence of prior papilloma growth, the T7-PKCεmice started developing carcinomas between 11 and 12 weeks of tumorpromotion in mice treated with DMBA and TPA. The appearance of theT7-PKCε mice resembled mice in a complete carcinogenesis experiment.Additionally, a few of the T7-PKCε mice initiated with DMBA and promotedwith acetone developed carcinomas.

The reduction in the T7-PKCε levels in the carcinomas indicates thatelevated levels of PKCε are not necessary for the maintenance of thecarcinoma. However, the positive correlation between elevated levels ofPKCε and carcinoma formation indicates that PKCε can induce themolecular changes necessary for carcinoma formation after treatment ofthe skin with a single dose of DMBA alone or in conjunction withrepeated TPA treatments.

Carcinoma development and metastatic malignant progression of transgenicmice. The T7-PKCε mouse line 215, which expressed T7-PKCε proteinapproximately 18-fold over endogenous PKCε levels, was further evaluatedas above for the development of carcinomas by the DMBA-TPA tumorpromotion protocol. At the beginning of the experiment, the 7-9 week oldmice exhibited no phenotypic abnormalities. In this experiment, femalewild-type and T7-PKCε transgenic mice were treated topically on shavedbacks with 100 nmol of DMBA in 0.2 ml acetone. Beginning two weekslater, 0.2 ml acetone or 5 nmol TPA in 0.2 ml acetone was appliedtwice-weekly to the dorsal skin. The number of mice for each group wasas follows: DMBA+TPA, wild-type mice, 15, T7-PKCε mice, 15;DMBA+acetone, wild-type mice, 14, T7-PKCε mice, 15.

The tumor incidence and multiplicity were observed weekly starting at 8weeks of TPA promotion. Carcinomas were recorded by gross observation asdownward-invading lesion Carcinoma bearing mice were observed forabnormal tumor growth in the lymph nodes. Wild-type and T7-PKCε micethat were positive for carcinoma formation in the preceding experimentwere sacrificed one week after the last treatment with TPA or acetone.

Treatment with TPA for 23 weeks elicited an average of 12 papillomas perwild-type mouse. However, in accordance with the previous findings, thetransgenic mice averaged less than 1 papilloma per mouse. The papillomasthat developed in transgenic mice were also much smaller than wild-typepapillomas.

In spite of the low papilloma burden, the transgenic mice developedcarcinoma independently of papilloma development. After 23 weeks oftumor promotion, 6 out of 15 (4%) transgenic mice were evaluated bygross examination as having at least one carcinoma, compared to 1 out of15 (7%) of the wild-type mice. Wild-type carcinomas developed fromexisting papillomas. Additionally, 3 out of 15 transgenic mice treatedwith DMBA+acetone also developed papilloma-independent carcinomas.Wild-type mice treated with DMBA+acetone developed no papillomas.

Because the treatment parameters were identical between this experimentand the prior experiment, the experimental data were normalized andcombined to determine whether the development of carcinomas in T7-PKCεafter DMBA initiation alone was statistically significant. Using theMSTAT computer program, provided by Dr. Norman Drinkwater (available forfree download at http://mcardle.oncology.wisc.edu/Mstat/), initiation byDMBA was shown to be sufficient for squamous cell carcinoma developmentin the transgenic mice. Two-sided p-values were calculated for tumor andmetastasis multiplicity by the Wilcoxon rank sum test. Two-sidedp-values were calculated using Fisher's exact test to compare tumorincidence. From the combined data, we determined that DMBA+acetonetreatment elicited carcinoma development in 7 out of 31 (22%) T7-PKCεmice, while wild-type mice never developed carcinomas. From the analysisof the combined data, we conclude that DMBA+acetone is sufficient toinduce carcinoma development in T7-PKCε mice.

Surprisingly, transgenic mice rapidly developed tumors in regional lymphnodes within three weeks after positive identification of carcinomas bygross observation. Three out of six mice positive for carcinomas alsocontained regional lymph nodes that bore tumors. The transgenic micethat developed carcinomas with DMBA+acetone treatment did not haveevidence of enlarged lymph nodes. However, the positive identificationof the carcinomas was less than three weeks before the mice weresacrificed at the conclusion of the experiment.

Tissues to be examined were excised promptly after euthanasia andimmediately placed in 10% neutral buffered formalin. Regional lymphnodes with evidence of tumor growth by gross observation were isolated,along with apparently normal lymph node in the same animal. Normaltissue was fixed for 1 hour and carcinomas and lymph nodes were fixedfor 2 to 3 hours in the formalin and then embedded in paraffin. 4 mmsections were cut for hematoxylin and eosin staining or immunostaining.Carcinomas were examined by a pathologist. Previous studies demonstratedconcordance between gross classification of skin tumors (papilloma vs.carcinoma) and subsequent microscopic evaluation by a pathologist.

By gross observation, both wild-type mouse and T7-PKCε mouse carcinomaswere identified by dark red color or the presence of blood clot on theskin surface. As the lesions progressed, necrosis occurred on thesurface of the cancer and surface ulceration resulted. Microscopically,cancer cells were identified by the presence of large pleiomorphicnuclei with prominent nucleoli and frequent mitoses. Areas ofintracellular keratinization and focal extracellular keratin depositswere identified. The cell cytoplasm was abundant and the cell surfaceexhibited intercellular bridges. Neutrophils were identified focallyadjacent to keratin pearls. The tumors from transgenic mice weremoderately differentiated squamous cell carcinoma (MDSC) based on asmall number of focal areas with typical squamous epithelium keratinformation, and a large number of areas composed of largelyundifferentiated cells. In histological sections of MDSC from twotransgenic mice initiated with DMBA and treated for 23 weeks with 5 nmolTPA, malignant cells were seen streaming from the hair follicle, oftenin the region of the sebaceous gland. This process often involvedmultiple adjacent hair follicles.

DMBA-initiated mice that had been treated for only 8 weeks with TPA oracetone were also harvested to determine the origin of premalignantlesions. After 8 weeks of TPA treatment, T7-PKCε mice displayed focalareas of increased hair follicle width, epidermal hyperplasia, andhyperkeratosis. Possible premalignant lesions were identified arisingfrom hair follicles; these lesions had cells with enlarged nuclei andprominent nucleoli and showed outward expansion from the hair follicle.

Multiple T7-PKCε transgenic mice exhibited enlarged regional lymph nodesafter the identification of primary tumor. The lymphoid tissue,identified by the presence of numerous collections ofwell-differentiated lymphocytic cells, was infiltrated by squamous cellcarcinoma. The carcinoma cells ranged in appearance fromundifferentiated clusters of epithelial cells to well differentiatedsquamous cells producing keratin (“keratin pearls”). Other areas showedundifferentiated carcinoma cells with numerous mitoses identified. Thecell morphology was identical to that seen in the primary cancer.

In contrast to T7-PKCε MDSC, the carcinomas of wild-type mice wereclassified as well differentiated squamous cell carcinoma (WDSC) basedon the observation that the majority of the tumor cells had a squamousappearance with abundant keratin formation. Microscopically, extensiveareas of intracellular keratinization and focal extracellular keratindeposits were identified. Malignant cells were observed streaming fromthe epidermis of the papilloma, not from the hair follicle. Epidermalhyperplasia was not observed in the uninvolved skin of wild-type mice.

All mice were harvested one week after the last TPA or acetonetreatment; therefore, the common transient effects of TPA treatment onmouse skin, including epidermal hyperplasia and keratinization, shouldmostly have subsided. This was the case with the uninvolved skin ofwild-type mice, which displayed no abnormalities. However, theuninvolved skin of T7-PKCε mice one week after TPA treatment stillexhibited hyperplasia of all epidermal cell layers with minimalhyperkeratosis, and small isolated foci of lymphocytic infiltrates wereidentified within the dermis.

T7-PKCε expressing transgenic mice display almost no papillomadevelopment during treatment with the two-stage DMBA+TPA tumor promotionprotocol in comparison to wild-type littermates. However, carcinomadevelopment appears to be enhanced compared to wild-type mice. In aneffort to better understand the origin and development ofpapilloma-independent carcinomas, T7-PKCε mice were further evaluatedfor the development of carcinomas by the DMBA+TPA tumor promotionprotocol.

Histopathological analysis of multiple T7-PKCε mice indicated thatsquamous cell carcinoma of T7-PKCε mice invaded the dermal region fromthe hair follicle. The squamous cell carcinoma of T7-PKCε mice rapidlymetastasized to regional lymph nodes as soon as 3 weeks after positiveidentification of carcinoma by gross observation. The tumors fromT7-PKCε transgenic mice were classified as moderately differentiatedsquamous cell carcinoma (MDSC). By comparison, the carcinomas ofwild-type mice, which appeared to originate from the interfollicularepidermis of papillomas, were classified as well differentiated squamouscell carcinoma (WDSC). WDSC derived from papillomas invaded the dermalarea with no evidence of metastatic progression.

The present invention is not intended to be limited to the foregoing,but to include all such variations and modifications as come within thescope of the appended claims.

6 1 2350 DNA Homo sapiens 1 aagcttatat tccatgctag ggttctggtg ttggtgcgtggggttggggt gggactgcag 60 aagtgccttt taagattatg tgattgactg atctgtcattggttccctgc catctttatc 120 ttttggattc ccctcggagg aggggaggaa ggagtttcttttgggtttta ttgaatcaaa 180 tgaaagggaa agtagaggtg ttcctatgga ggggaggaaggagtttcttt tgggttttat 240 tgaatcaaat gaaagggaaa gtagaggtgt tcctatgtcccgggctccgg agcttctatt 300 cctgggccct gcataagaag gagacatggt ggtggtggtggtgggtgggg gtggtggggc 360 acagaggaag ccgatgctgg gctctgcacc ccattcccgctcccagatcc ctctggatat 420 agcaccccct ccagtgagca cagcctcccc ttgccccacagccaacagca acatgcctcc 480 caacaaagca tctgtccctc agccaaaacc cctgttgcctctctctgggg aaattgtagg 540 gctgggccag ggtgggggga ccattctctg cagggagattaggagtgtct gtcaggggcg 600 ggtggagcgg ggtggggccc tggcttactc acatccttgagagtcctttg ctggcagatt 660 tggggagccc acagctcaga tgtctgtctc agcattgtcttccaagctcc taggccacag 720 tagtggggcg ctcccttctc tggcttcttc tttggtgacagtcaaggtgg ggttgggggt 780 gacgaagggt cctgcttctc ttctaggagc agttgatcccaggaagagca ttggagcctc 840 cagcaggggc tgttggggcc tgtctgagga gataggatgcgtcaggcagc cccagacacg 900 atcacattcc tctcaacatg cctgccgggg tctgtggagccgaggggctg atgggagggt 960 ggggtggggg ccggaagggt ttgctttggg aggttgtctgggagattgct gaagttttga 1020 tatacacacc tccaaagcag gaccaagtgg actcctagaaatgtcccctg acccttgggg 1080 cttcaggagt cagggaccct cgtgtccacc tcagccttgcccttgcacag cccagctcca 1140 ctccagcctc tactcctccc cagaacatct cctgggccagttccacaagg ggctcaaacg 1200 agggcacctg agctgcccac actagggatg ttctgggggtctgagaagat atctggggct 1260 ggaagaataa aaggcccccc taggcctgtt cctggatgcagctccagcca ctttggggct 1320 aagcctgggc aataacaatg ccaacgaggc ttcttgccatactcggttta caaaaccctt 1380 tacatacatt gtcgcattgg attctcagag ctgactgcactaagcagaat agatggtatg 1440 actcccactt tgcagatgag aacactgagg ctcagagaagtgcgaagccc tgggtcacag 1500 aggcgtaaat gcagagccag gacccacctg aagacccacctgactccagg atgtttcctg 1560 cctccatgag gccacctgcc ctatggtgtg gtggatgtgagatcctcacc atagggagga 1620 gattagggtc tgtgctcagg gctggggaga ggtgcctggatttctctttg atggggatgt 1680 tggggtggga atcacgatac acctgatcag ctgggtgtatttcagggatg gggcagactt 1740 ctcagcacag cacggcaggt caggcctggg agggccccccagacctcctt gtctctaata 1800 gagggtcatg gtgagggagg cctgtctgtg cccaaggtgaccttgccatg ccggtgcttt 1860 ccagccgggt atccatcccc tgcagcagca ggcttcctctacgtggatgt taaaggccca 1920 ttcagttcat ggagagctag caggaaacta ggtttaaggtgcagaggccc tgctctctgt 1980 caccctggct aagcccagtg cgtgggttcc tgagggctgggactcccagg gtccgatggg 2040 aaagtgtagc ctgcaggccc acacctcccc ctgtgaatcacgcctggcgg gacaagaaag 2100 cccaaaacac tccaaacaat gagtttccag taaaatatgacagacatgat gaggcggatg 2160 agaggaggga cctgcctggg agttggcgct agcctgtgggtgatgaaagc caaggggaat 2220 ggaaagtgcc agacccgccc cctacccatg agtataaagcactcgcatcc ctttgcaatt 2280 tacccgagca ccttctcttc actcagcctt ctgctcgctcgctcacctcc ctcctctgca 2340 ccatgactac 2350 2 750 DNA Mus musculus 2tggcaagaag gtgctggctg ccttcagtga gggtctgagt cacctggaca acctcaaagg 60cacctttgct aagctgagtg aactgcactg tgacaagctg cacgtggatc ctgagaactt 120cagggtgagt ttggggaccc ttgattgttc tttctttttc gctattgtaa aattcatgtt 180atatggaggg ggcaaagttt tcagggtgtt gtttagaatg ggaagatgtc ccttgtatca 240ccatggaccc tcatgataat tttgtttctt tcactttcta ctctgttgac aaccattgtc 300tcctcttatt ttcttttcat tttctgtaac tttttcgtta aactttagct tgcatttgta 360acgaattttt aaattcactt ttgtttattt gtcagattgt aagtactttc tctaatcact 420tttttttcaa ggcaatcagg gtatattata ttgtacttca gcacagtttt agagaacaat 480tgttataatt aaatgataag gtagaatatt tctgcatata aattctggct ggcgtggaaa 540tattcttatt ggtagaaaca actacatcct ggtcatcatc ctgcctttct ctttatggtt 600acaatgatat acactgtttg agatgaggat aaaatactct gagtccaaac cgggcccctc 660tgctaaccat gttcatgcct tcttcttttt cctacagctc ctgggcaacg tgctggttat 720tgtgctgtct catcattttg gcaaagaatt 750 3 2274 DNA Artificial SequenceDescription of Artificial Sequence T7 tag and mouse protein kinase Cepsilon coding sequence 3 atggctagca tgactggtgg acagcaaatg ggtcggatccgaattcgagc tccgtcgacc 60 atg gta gtg ttc aat ggc ctt ctt aag atc aaa atctgc gag gcg gtg 108 Met Val Val Phe Asn Gly Leu Leu Lys Ile Lys Ile CysGlu Ala Val 1 5 10 15 agc ttg aag ccc aca gcc tgg tcg ctg cgc cat gcggtg gga ccc cgg 156 Ser Leu Lys Pro Thr Ala Trp Ser Leu Arg His Ala ValGly Pro Arg 20 25 30 cca cag acg ttc ctt ttg gac ccc tac att gcc ctt aacgtg gac gac 204 Pro Gln Thr Phe Leu Leu Asp Pro Tyr Ile Ala Leu Asn ValAsp Asp 35 40 45 tcg cgc atc ggc caa aca gcc acc aag caa aag acc aac agcccg gcc 252 Ser Arg Ile Gly Gln Thr Ala Thr Lys Gln Lys Thr Asn Ser ProAla 50 55 60 tgg cac gat gag ttc gtc acc gat gtg tgc aat ggg cgc aag atcgag 300 Trp His Asp Glu Phe Val Thr Asp Val Cys Asn Gly Arg Lys Ile Glu65 70 75 80 ctg gct gtc ttt cac gac gct cct atc ggc tac gac gac ttc gtggcc 348 Leu Ala Val Phe His Asp Ala Pro Ile Gly Tyr Asp Asp Phe Val Ala85 90 95 aac tgc acc atc cag ttc gag gag ctg ctg cag aat ggg agc cgt cac396 Asn Cys Thr Ile Gln Phe Glu Glu Leu Leu Gln Asn Gly Ser Arg His 100105 110 ttc gag gac tgg att gac ctg gag cca gaa gga aaa gtg tac gtg atc444 Phe Glu Asp Trp Ile Asp Leu Glu Pro Glu Gly Lys Val Tyr Val Ile 115120 125 atc gat ctc tcg gga tca tcg ggt gaa gcc cct aaa gac aat gaa gaa492 Ile Asp Leu Ser Gly Ser Ser Gly Glu Ala Pro Lys Asp Asn Glu Glu 130135 140 cga gtg ttc agg gag cgt atg cgg cca agg aag cgg caa ggg gct gtc540 Arg Val Phe Arg Glu Arg Met Arg Pro Arg Lys Arg Gln Gly Ala Val 145150 155 160 agg cgc agg gtc cac cag gtc aat ggc cac aag ttc atg gcc acctac 588 Arg Arg Arg Val His Gln Val Asn Gly His Lys Phe Met Ala Thr Tyr165 170 175 ttg cgg caa ccc acc tac tgc tcc cac tgc aga gat ttc atc tggggt 636 Leu Arg Gln Pro Thr Tyr Cys Ser His Cys Arg Asp Phe Ile Trp Gly180 185 190 gtc ata gga aaa cag gga tat caa tgt caa gtt tgc act tgc gttgtc 684 Val Ile Gly Lys Gln Gly Tyr Gln Cys Gln Val Cys Thr Cys Val Val195 200 205 cac aag cga tgt cat gag ctc att att aca aag tgc gct ggg ctgaag 732 His Lys Arg Cys His Glu Leu Ile Ile Thr Lys Cys Ala Gly Leu Lys210 215 220 aaa cag gaa acc cct gac gag gtg ggc tcc caa cgg ttc agc gtcaac 780 Lys Gln Glu Thr Pro Asp Glu Val Gly Ser Gln Arg Phe Ser Val Asn225 230 235 240 atg ccc cac aag ttc ggg atc cac aac tac aag gtc ccc acgttc tgt 828 Met Pro His Lys Phe Gly Ile His Asn Tyr Lys Val Pro Thr PheCys 245 250 255 gac cac tgt ggg tcc ctg ctc tgg ggc ctc ttg cgg cag ggcttg cag 876 Asp His Cys Gly Ser Leu Leu Trp Gly Leu Leu Arg Gln Gly LeuGln 260 265 270 tgt aaa gtc tgc aaa atg aat gtt cac cgg cga tgt gag accaac gtg 924 Cys Lys Val Cys Lys Met Asn Val His Arg Arg Cys Glu Thr AsnVal 275 280 285 gct ccc aac tgt ggg gta gac gcc aga gga att gcc aaa gtgctg gct 972 Ala Pro Asn Cys Gly Val Asp Ala Arg Gly Ile Ala Lys Val LeuAla 290 295 300 gac ctc ggt gtt act cca gac aaa atc acc aac agt ggc caaagg agg 1020 Asp Leu Gly Val Thr Pro Asp Lys Ile Thr Asn Ser Gly Gln ArgArg 305 310 315 320 aaa aag ctc gct gct ggt gct gag tcc cca cag ccg gcttct gga aac 1068 Lys Lys Leu Ala Ala Gly Ala Glu Ser Pro Gln Pro Ala SerGly Asn 325 330 335 tcc cca tct gaa gac gac cga tcc aag tca gcg ccc acctcc cct tgt 1116 Ser Pro Ser Glu Asp Asp Arg Ser Lys Ser Ala Pro Thr SerPro Cys 340 345 350 gac cag gaa cta aaa gaa ctt gaa aac aac atc cgg aaggcc ttg tca 1164 Asp Gln Glu Leu Lys Glu Leu Glu Asn Asn Ile Arg Lys AlaLeu Ser 355 360 365 ttt gac aac cga gga gag gag cac cga gcg tcg tcg gccacc gat ggc 1212 Phe Asp Asn Arg Gly Glu Glu His Arg Ala Ser Ser Ala ThrAsp Gly 370 375 380 cag ctg gca agc ccc gga gag aat ggg gaa gtc cgg ccaggc cag gcc 1260 Gln Leu Ala Ser Pro Gly Glu Asn Gly Glu Val Arg Pro GlyGln Ala 385 390 395 400 aag cgc ttg ggg ctg gat gag ttc aac ttc atc aaagtg ttg ggc aaa 1308 Lys Arg Leu Gly Leu Asp Glu Phe Asn Phe Ile Lys ValLeu Gly Lys 405 410 415 ggc agc ttt ggc aag gtc atg ttg gcg gaa ctc aaaggc aaa gat gaa 1356 Gly Ser Phe Gly Lys Val Met Leu Ala Glu Leu Lys GlyLys Asp Glu 420 425 430 gtc tac gct gtg aag gtc ttg aag aag gac gtt atccta caa gac gat 1404 Val Tyr Ala Val Lys Val Leu Lys Lys Asp Val Ile LeuGln Asp Asp 435 440 445 gat gtg gac tgc aca atg aca gag aag agg att ttggct ctg gct cgg 1452 Asp Val Asp Cys Thr Met Thr Glu Lys Arg Ile Leu AlaLeu Ala Arg 450 455 460 aaa cac cct tat cta acc caa ctc tat tgc tgc ttccag acc aag gac 1500 Lys His Pro Tyr Leu Thr Gln Leu Tyr Cys Cys Phe GlnThr Lys Asp 465 470 475 480 cgc ctc ttc ttc gtc atg gaa tat gta aat ggtgga gac ctc atg ttc 1548 Arg Leu Phe Phe Val Met Glu Tyr Val Asn Gly GlyAsp Leu Met Phe 485 490 495 cag att cag cgg tcc cga aaa ttt gat gag cctcgt tct cgg ttc tat 1596 Gln Ile Gln Arg Ser Arg Lys Phe Asp Glu Pro ArgSer Arg Phe Tyr 500 505 510 gcc gca gag gtc aca tcg gcc ctc atg ttt ctccac cag cat gga gtg 1644 Ala Ala Glu Val Thr Ser Ala Leu Met Phe Leu HisGln His Gly Val 515 520 525 atc tac agg gat ttg aaa ctg gac aac atc cttcta gat gca gaa ggc 1692 Ile Tyr Arg Asp Leu Lys Leu Asp Asn Ile Leu LeuAsp Ala Glu Gly 530 535 540 cac tgc aag ctg gct gac ttt ggg atg tgc aaggaa ggg att atg aat 1740 His Cys Lys Leu Ala Asp Phe Gly Met Cys Lys GluGly Ile Met Asn 545 550 555 560 ggt gtg aca act acc acc ttc tgt ggg actcct gac tac ata gct cca 1788 Gly Val Thr Thr Thr Thr Phe Cys Gly Thr ProAsp Tyr Ile Ala Pro 565 570 575 gag atc cta cag gag ttg gag tac ggc ccctca gtg gac tgg tgg gcc 1836 Glu Ile Leu Gln Glu Leu Glu Tyr Gly Pro SerVal Asp Trp Trp Ala 580 585 590 ctg gga gtg ctg atg tac gag atg atg gctggg cag ccc ccc ttt gaa 1884 Leu Gly Val Leu Met Tyr Glu Met Met Ala GlyGln Pro Pro Phe Glu 595 600 605 gct gac aac gag gac gac ttg ttc gaa tccatc ctt cat gat gat gtt 1932 Ala Asp Asn Glu Asp Asp Leu Phe Glu Ser IleLeu His Asp Asp Val 610 615 620 ctc tat cct gtc tgg ctc agc aag gaa gctgtc agc atc ctg aaa gct 1980 Leu Tyr Pro Val Trp Leu Ser Lys Glu Ala ValSer Ile Leu Lys Ala 625 630 635 640 ttc atg acc aag aac ccg cac aag cgcctg ggc tgt gtg gca gcg cag 2028 Phe Met Thr Lys Asn Pro His Lys Arg LeuGly Cys Val Ala Ala Gln 645 650 655 aac ggg gag gac gcc atc aag caa catcca ttc ttc aag gag att gac 2076 Asn Gly Glu Asp Ala Ile Lys Gln His ProPhe Phe Lys Glu Ile Asp 660 665 670 tgg gta ctg ctg gag cag aag aaa atcaag ccc ccc ttc aag ccg aga 2124 Trp Val Leu Leu Glu Gln Lys Lys Ile LysPro Pro Phe Lys Pro Arg 675 680 685 att aaa acc aaa aga gat gtc aat aacttt gac caa gac ttt acg cgg 2172 Ile Lys Thr Lys Arg Asp Val Asn Asn PheAsp Gln Asp Phe Thr Arg 690 695 700 gaa gag cca ata ctt aca ctt gtg gatgaa gca atc att aag cag atc 2220 Glu Glu Pro Ile Leu Thr Leu Val Asp GluAla Ile Ile Lys Gln Ile 705 710 715 720 aac cag gaa gaa tty aaa ggc ttctcc tac ttt ggt gaa gac ctg atg 2268 Asn Gln Glu Glu Phe Lys Gly Phe SerTyr Phe Gly Glu Asp Leu Met 725 730 735 ccc tga 2274 Pro 4 737 PRTArtificial Sequence Description of Artificial Sequence T7 tag and 4 MetVal Val Phe Asn Gly Leu Leu Lys Ile Lys Ile Cys Glu Ala Val 1 5 10 15Ser Leu Lys Pro Thr Ala Trp Ser Leu Arg His Ala Val Gly Pro Arg 20 25 30Pro Gln Thr Phe Leu Leu Asp Pro Tyr Ile Ala Leu Asn Val Asp Asp 35 40 45Ser Arg Ile Gly Gln Thr Ala Thr Lys Gln Lys Thr Asn Ser Pro Ala 50 55 60Trp His Asp Glu Phe Val Thr Asp Val Cys Asn Gly Arg Lys Ile Glu 65 70 7580 Leu Ala Val Phe His Asp Ala Pro Ile Gly Tyr Asp Asp Phe Val Ala 85 9095 Asn Cys Thr Ile Gln Phe Glu Glu Leu Leu Gln Asn Gly Ser Arg His 100105 110 Phe Glu Asp Trp Ile Asp Leu Glu Pro Glu Gly Lys Val Tyr Val Ile115 120 125 Ile Asp Leu Ser Gly Ser Ser Gly Glu Ala Pro Lys Asp Asn GluGlu 130 135 140 Arg Val Phe Arg Glu Arg Met Arg Pro Arg Lys Arg Gln GlyAla Val 145 150 155 160 Arg Arg Arg Val His Gln Val Asn Gly His Lys PheMet Ala Thr Tyr 165 170 175 Leu Arg Gln Pro Thr Tyr Cys Ser His Cys ArgAsp Phe Ile Trp Gly 180 185 190 Val Ile Gly Lys Gln Gly Tyr Gln Cys GlnVal Cys Thr Cys Val Val 195 200 205 His Lys Arg Cys His Glu Leu Ile IleThr Lys Cys Ala Gly Leu Lys 210 215 220 Lys Gln Glu Thr Pro Asp Glu ValGly Ser Gln Arg Phe Ser Val Asn 225 230 235 240 Met Pro His Lys Phe GlyIle His Asn Tyr Lys Val Pro Thr Phe Cys 245 250 255 Asp His Cys Gly SerLeu Leu Trp Gly Leu Leu Arg Gln Gly Leu Gln 260 265 270 Cys Lys Val CysLys Met Asn Val His Arg Arg Cys Glu Thr Asn Val 275 280 285 Ala Pro AsnCys Gly Val Asp Ala Arg Gly Ile Ala Lys Val Leu Ala 290 295 300 Asp LeuGly Val Thr Pro Asp Lys Ile Thr Asn Ser Gly Gln Arg Arg 305 310 315 320Lys Lys Leu Ala Ala Gly Ala Glu Ser Pro Gln Pro Ala Ser Gly Asn 325 330335 Ser Pro Ser Glu Asp Asp Arg Ser Lys Ser Ala Pro Thr Ser Pro Cys 340345 350 Asp Gln Glu Leu Lys Glu Leu Glu Asn Asn Ile Arg Lys Ala Leu Ser355 360 365 Phe Asp Asn Arg Gly Glu Glu His Arg Ala Ser Ser Ala Thr AspGly 370 375 380 Gln Leu Ala Ser Pro Gly Glu Asn Gly Glu Val Arg Pro GlyGln Ala 385 390 395 400 Lys Arg Leu Gly Leu Asp Glu Phe Asn Phe Ile LysVal Leu Gly Lys 405 410 415 Gly Ser Phe Gly Lys Val Met Leu Ala Glu LeuLys Gly Lys Asp Glu 420 425 430 Val Tyr Ala Val Lys Val Leu Lys Lys AspVal Ile Leu Gln Asp Asp 435 440 445 Asp Val Asp Cys Thr Met Thr Glu LysArg Ile Leu Ala Leu Ala Arg 450 455 460 Lys His Pro Tyr Leu Thr Gln LeuTyr Cys Cys Phe Gln Thr Lys Asp 465 470 475 480 Arg Leu Phe Phe Val MetGlu Tyr Val Asn Gly Gly Asp Leu Met Phe 485 490 495 Gln Ile Gln Arg SerArg Lys Phe Asp Glu Pro Arg Ser Arg Phe Tyr 500 505 510 Ala Ala Glu ValThr Ser Ala Leu Met Phe Leu His Gln His Gly Val 515 520 525 Ile Tyr ArgAsp Leu Lys Leu Asp Asn Ile Leu Leu Asp Ala Glu Gly 530 535 540 His CysLys Leu Ala Asp Phe Gly Met Cys Lys Glu Gly Ile Met Asn 545 550 555 560Gly Val Thr Thr Thr Thr Phe Cys Gly Thr Pro Asp Tyr Ile Ala Pro 565 570575 Glu Ile Leu Gln Glu Leu Glu Tyr Gly Pro Ser Val Asp Trp Trp Ala 580585 590 Leu Gly Val Leu Met Tyr Glu Met Met Ala Gly Gln Pro Pro Phe Glu595 600 605 Ala Asp Asn Glu Asp Asp Leu Phe Glu Ser Ile Leu His Asp AspVal 610 615 620 Leu Tyr Pro Val Trp Leu Ser Lys Glu Ala Val Ser Ile LeuLys Ala 625 630 635 640 Phe Met Thr Lys Asn Pro His Lys Arg Leu Gly CysVal Ala Ala Gln 645 650 655 Asn Gly Glu Asp Ala Ile Lys Gln His Pro PhePhe Lys Glu Ile Asp 660 665 670 Trp Val Leu Leu Glu Gln Lys Lys Ile LysPro Pro Phe Lys Pro Arg 675 680 685 Ile Lys Thr Lys Arg Asp Val Asn AsnPhe Asp Gln Asp Phe Thr Arg 690 695 700 Glu Glu Pro Ile Leu Thr Leu ValAsp Glu Ala Ile Ile Lys Gln Ile 705 710 715 720 Asn Gln Glu Glu Phe LysGly Phe Ser Tyr Phe Gly Glu Asp Leu Met 725 730 735 Pro 5 598 DNA Homosapiens 5 tcaggcctag gaggcccccc gtgtggacac agatcccact ggaagatcccctctcctgcc 60 caagcacttc acagctggac cctgcttcac cctcaccccc tcctggcaatcaatacagct 120 tcattatctg agttgcataa ttctcgcctc tctctggtca ttgttaggagtgggggtggg 180 gagaaagtgg gagagcatct ctttggagct tgtcatgcac ctggctatggcccctgggac 240 tgggagaaaa gtcctggggg tgggttgggc tcaggtccca ggatatctttcgccatctca 300 gaagacacag atagatgtgt gtaccaggtc atatgtggtg tctcctagggtacggaggga 360 tattcattca tttactcact cattttcatg tgtgtccatt cattcaccagatattgagtg 420 cctctatgtc aggcactatg ttaggttaag gattcctgat gtttttgtgtatcagggatt 480 ccttggagaa tattgaaagc tatagatctt tccttctgcc ccctaccttcaaataagcat 540 acatacattt gcatacatgt catggggttc atgggtctcc tagagctccttaccggag 598 6 9 PRT Mus musculus 6 Glu Arg Lys Arg Thr Leu Arg Arg Leu1 5

We claim:
 1. A genetically modified FVB/N mouse having epidermal cellsthat comprise a protein kinase Cε activity higher than that of wild-typeFVB/N epidermal cells.
 2. An FVB/N mouse as claimed in claim 1 whereinthe protein kinase Cε level is at least 5-fold higher than that ofwild-type FVB/N epidermal cells.
 3. An FVB/N mouse as claimed in claim 1wherein the protein kinase Cε level is at least 12-fold higher than thatof wild-type FVB/N epidermal cells.
 4. An FVB/N mouse as claimed inclaim 1 wherein the protein kinase Cε level is at least 15-fold higherthan that of wild-type FVB/N epidermal cells.
 5. An FVB/N mouse asclaimed in claim 1 wherein the protein kinase Cε level is at least18-fold higher than that of wild-type FVB/N epidermal cells.
 6. An FVB/Nmouse as claimed in claim 1 wherein the mouse exhibits metastaticsquamous cell carcinoma disease.
 7. An FVB/N mouse as claimed in claim 1comprising in the epidermal cells a transgene that comprises apolynucleotide that encodes Protein Kinase Cε under the transcriptionalcontrol of an upstream promoter active in keratinocytes and a downstreampolyA addition sequence.
 8. An FVB/N mouse as claimed in claim 7 whereinthe upstream promoter is a keratin promoter.
 9. An FVB/N mouse asclaimed in claim 8 wherein the upstream promoter is a K14 promoter. 10.An FVB/N mouse as claimed in claim 7 further comprising a transcriptionenhancing element.
 11. An FVB/N mouse as claimed in claim 10 wherein thetranscription enhancing element is a β-globin intron sequence.
 12. AnFVB/N mouse as claimed in claim 7 wherein the transgene comprises SEQ IDNO:3.
 13. A transgenic mouse whose somatic and germ cells contain achimeric DNA sequence comprising a keratin promoter/regulatory sequenceoperably linked to a sequence encoding protein kinase Cε, whereinepidermal expression of said protein kinase Cε results in a phenotypeselected from the group consisting of inflammation, hyperkeratosis,hyperplasia, cellular hypertrophy, and ulceration.
 14. An FVB/N mouse asclaimed in claim 1 wherein the mouse is suppressed for papillomaproduction and susceptible to developing squamous cell carcinoma aftertumor in initiation and promotion.
 15. An FVB/N mouse as claimed inclaim 14 wherein the mouse is further susceptible to developingmetastatic squamous cell carcinoma after tumor initiation and promotion.16. A method for inducing squamous cell carcinoma in an FVB/N mousesuppressed for formation of papillomas, the method comprising the stepsof: treating an FVB/N mouse having epidermal cells that comprise aprotein kinase Cε activity higher than that of wild-type FVB/N epidermalcells with a tumor initiating agent; and treating the initiated mouserepeatedly with a tumor promoting agent until the mouse developssquamous cell carcinoma while suppressing formation of papillomas.
 17. Amethod as claimed in claim 16 wherein the tumor initiating agent is7,12-dimethylbenz[a]anthracene and the tumor promoting agent is12-O-tetradecanoylphorbol-13-acetate.
 18. A method as claimed in claim16 wherein the protein kinase Cε activity is at least 12-fold higherthan that of wild-type FVB/N epidermal cells and wherein the squamouscell carcinoma is metastatic.
 19. A method as claimed in claim 18wherein the protein kinase Cε activity is at least 15-fold higher thanthat of wild-type FVB/N epidermal cells.
 20. A method as claimed inclaim 18 wherein the protein kinase Cε activity is at least 18-foldhigher than that of wild-type FVB/N epidermal cells.
 21. A method asclaimed in claim 16 wherein the epidermal cells of the FVB/N mousecomprise a polynucleotide that encodes Protein Kinase Cε under thetranscriptional control of an upstream promoter active in keratinocytesand a downstream polyA addition sequence.
 22. A method for evaluatingeffectiveness of a putative agent as a chemopreventative againstsquamous cell carcinoma disease in a mammal, the method comprising thesteps of: administering at least one putative agent topapilloma-suppressed FVB/N mice having epidermal cells that comprise aprotein kinase Cε activity higher than that of wild-type FVB/N epidermalcells, wherein the mice are susceptible to squamous cell carcinomadisease after tumor initiation and promotion, and wherein the agent isadministered in an amount effective to prevent onset of the disease;treating the mice with a tumor initiating agent; and treating theinitiated mice repeatedly with a tumor promoting agent evaluating theeffectiveness of the at least one putative agent; and selecting from theat least one putative agent an effective chemopreventative agent.